Zymo CleanUp
Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
Transfer into the Zymo column (small clear guys)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction

Then perform PCA2:

''Amplification'''
Now, you need to do an amplification of the correct full-length chunks. Clean up the assembly reaction with a zymo column; don't bother running it on a gel - it'll be a smeary mess and won't really help you. Save the purified product in case this step fails! For the amplification reaction, do a normal phusion program with 1 ul of the cleaned up assembly reaction as template, and using the outermost oligos for the chunk. That is:
'''Recipe'''
1 ul each outer oligo (10 uM)
-Dilute F/R oligos 1:10 from 100uM; in this case, oligo CCOMT1-12/CCOMT1-15
1 ul purified pca product
.5 ul phusion
10 ul 5x phusion buffer
5 ul 2mM dNTPs
32.5 ul H2O
Samples given to John to run the Program. 1:10 Oligo12/Oligo15 dilutions in small PCR tubes. Also stored purified PCA1 product in 4o box.
'''Program'''
2 min initial denature at 94oC
30 sec denature at 94oC
30 sec anneal at 60oC [This should be high, as your outer oligos now have a huge overlap with the correct product]
30 sec extension at 68oC
repeat 2-4 30 times total

"'3/14/2013
Gel from all class samples. Gel is of PCA2 products for various synthons.
Lane1=His-tag part's IPCR product,
Lane2=Molecular weight standards
Rest of the lanes are PCA2's

Zymo CleanUp on PCA2 product
Add 180 uL of Zymo ADB buffer (brown bottle) to a 33uL or 50uL reaction.
Transfer into the Zymo column (small clear guys)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer (which is basically 70% ethanol)
spin through, discard waste.
Add 200 uL of Zymo Wash Buffer
spin through, discard waste.
spin for 90 seconds, full speed to dry.
elute with water into a fresh Eppendorf tube, use the same volume of water as the volume of the original reaction
EcoRI/BamHI Digest on PCA2
1. 1uL of NEB Buffer 2 (ADD THIS FIRST)
2. 8uL of eluted PCR product
3. 0.5uL EcoRI
4. 0.5uL BamHI
Incubate at 37 degrees on the thermocycler for 1hr
Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point
If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column

6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL Vector digest
1uL Insert digest
0.5uL T4 DNA Ligase
Pound upside down on the bench to mix
Give it a quick spin to send it back to the bottom of the tube
Incubate on the benchtop for 30min
Put on ice and proceed to the transformation

Transformation
Take competent cells to which TSS has been added.
Heat shock
Rescue with 2YT

Thaw a 200 uL aliquot of cells on ice
Add 50 uL of water to the cells (if greater volume is desired)
Add 30 uL of KCM to the cells
Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
Add 70 uL of the cell cocktail to the ligation, stir to mix
Let sit on ice for 10 min
Heat shock for 90 seconds at 42 (longer incubation may work better)
Put back on ice for 1 min
Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour (DID NOT RESCUE FOR 1 HOUR BECAUSE AMPICILLIN)
Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

'4/2/2013

Issue with colonies, could be issue with restrictive enzymes, buffers used, ran only a small amount of gel.
Therefore redid PCA2 pdt cleanup
Protocol
EcoRI/BglII Digest on PCA2
1. 1uL of NEB Buffer 2 (ADD THIS FIRST)
2. 8uL of eluted PCR product
3. 0.5uL EcoRI
4. 0.5uL BgIII
Incubate at 37 degrees on the thermocycler for 1hr
Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees. ****NOTE: If you are running short of time, this is an acceptable stopping point
If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column
In the meantime run gel purification on PCR pdt digestion
Zymo Gel Purification
1. Add 0.5 uL Loading Dye to 2uL sample
2. Load all of sample into well
Loaded to lane 4 (2nd gel)

Then did regular zymo clean up on the PCA2 that was digested with EcoRI/BglII
(added 8µL of water to eppendorf tube to elute)

4/4/2013
Redid what we did on 3/19 so
Ligation -> Heat Shock -> Plating

Ligation of EcoRI/BamHI digests

6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL Vector digest
1uL Insert digest
0.5uL T4 DNA Ligase
Pound upside down on the bench to mix
Give it a quick spin to send it back to the bottom of the tube
Incubate on the benchtop for 30min
Put on ice and proceed to the transformation

Transformation
Take competent cells to which TSS has been added.
Heat shock
Rescue with 2YT

Thaw a 200 uL aliquot of cells on ice
Add 50 uL of water to the cells (if greater volume is desired)
Add 30 uL of KCM to the cells
Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
Add 70 uL of the cell cocktail to the ligation, stir to mix
Let sit on ice for 10 min
Heat shock for 90 seconds at 42 (longer incubation may work better)
Put back on ice for 1 min
Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour (DID NOT RESCUE FOR 1 HOUR BECAUSE AMPICILLIN)
Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

"'4/9/13
Colonies did not quite work for me, but I'm going to try using one of the white colonies (there was only one white colony).
Did not do colony PCR, so only added 5mL of media to culture tubes (most people had 4, I only had 1). Picked out white colony with pipette tip then placed tip into culture tube.

4/11/2013

Miniprep purification of DNA
MINIPREP (2mL) Procedure for Plasmid DNA Purification
(using the QIAGEN QIAPrep Spin Miniprep kit)
!!!!! Make sure Ethanol has been added to the PE Buffer !!!!!
!!!!! Make sure that RNAse has been added to the P1 Buffer !!!!!
Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube. (Did this by first doing 1mL, spin, get rid of supernatant, add 1mL again, then spin again)
Dump supernatant
Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly
Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
Spin in centrifuge at top speed for 5 minutes.
Label blue columns with an alcohol-resistant lab pen.
Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
Wash each column with 500 uL of PB buffer.
Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
Wash with 750uL of PE buffer (washes the salts off the resins).
Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
Label new Microcentrifuge tubes and put columns in them.
Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
Spin in centrifuge at top speed for 30 seconds.
Take out columns and cap the tubes.
Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

Run an analytical gel
(Put 2µL of loading dye into the 10µL of reaction sample; then loaded 12µL into lane 6)
Take a picture of the gel
Calculate the expected fragment sizes
Are the calculated sizes consistent with the bands on the gel?
My sample did not work, so normally would have to keep trouble shooting till it works
Professor Anderson has already started the process over and is on the ligation step

4/16/2013

Again colony PCR did not work, but will analyze sequencing data from other people's colony PCR to see which one they will use.
Looked at Megan's asfb-1 sequencing data, and it worked.
Chris has been working on mine and is on the last ligation step (after PCA2), he will hopefully finish it at 17:00 today. Will then plate colonies tomorrow, on Thursday will hopefully be able to
do colony PCR and will be able to send out colonies for sequencing. Will then hopefully get sequencing data on Friday, then can start Golden Gate of combining Megan's and I's asbf part together. For the time being just watched what other people were doing.

4/18/2013
Redo what we did last week on 4/11/2013

Miniprep purification of DNA
MINIPREP (2mL) Procedure for Plasmid DNA Purification
(using the QIAGEN QIAPrep Spin Miniprep kit)
!!!!! Make sure Ethanol has been added to the PE Buffer !!!!!
!!!!! Make sure that RNAse has been added to the P1 Buffer !!!!!
Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube. (Did this by first doing 1mL, spin, get rid of supernatant, add 1mL again, then spin again)
Dump supernatant
Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly
Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
Spin in centrifuge at top speed for 5 minutes.
Label blue columns with an alcohol-resistant lab pen.
Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
Wash each column with 500 uL of PB buffer.
Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
Wash with 750uL of PE buffer (washes the salts off the resins).
Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
Label new Microcentrifuge tubes and put columns in them.
Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
Spin in centrifuge at top speed for 30 seconds.
Take out columns and cap the tubes.
Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

Then do restriction map w/ BsmBI

Set up the following 10uL reaction in a PCR tube:
6.5 uL ddH2O
2uL Miniprepped plasmid
1uL 10x NEB Buffer 2
0.5uL BsmBI
Incubate at 37 on the thermocycler for 30 minutes
Run an analytical gel
(Put 2µL of loading dye into the 10µL of reaction sample; then loaded 12µL into lane 6)
Take a picture of the gel
Calculate the expected fragment sizes
Are the calculated sizes consistent with the bands on the gel?
Ran gels and the only culture that appeared to work was culture (D). That sample was sent for sequencing, and hopefully tomorrow (4/19/2013), will have good results
to finally do golden-gate to form asbf part.
4/19/2013
Protocol for Golden Gate
6.5µL ddH2O to 10µL
1µL Ligase Buffer
0.5µL Ligase
0.5µL BsmBI
1µL of synthon mix (ASBF-1 and ASBF-2)
0.5µL Vector

4/23/2013
'Transformation
Take competent cells to which TSS has been added.
Heat shock
Rescue with 2YT

Thaw a 200 uL aliquot of cells on ice
Add 20 uL of KCM to the cells
Put your ligation mixture on ice, let cool a minute or two (for Miniprep product, dilute by 10, then use 1uL of dilution)
Add 70 uL of the cell cocktail to the ligation, stir to mix
Let sit on ice for 10 min
Heat shock for 90 seconds at 42 (longer incubation may work better)
Put back on ice for 1 min
Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour (NEED TO RESCUE FOR 1 HOUR BECAUSE CAM)
Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight

4/25/2013

Pellet 2 mL saturated culture by spinning full speed, 30 seconds in a 2mL Microcentrifuge tube. (Did this by first doing 1mL, spin, get rid of supernatant, add 1mL again, then spin again)
Dump supernatant
Add 250uL of P1 buffer into each tube. Resuspend the cells thoroughly
Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become clearer.
Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
Spin in centrifuge at top speed for 5 minutes.
Label blue columns with an alcohol-resistant lab pen.
Pour liquid into columns, and place the columns into the centrifuge. Spin at full speed for 15 seconds.
Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
Wash each column with 500 uL of PB buffer.
Spin in centrifuge at full speed for 15 seconds, then flick out the liquid again.
Wash with 750uL of PE buffer (washes the salts off the resins).
Spin in centrifuge at full speed for 15 seconds and flick out liquid again.
Spin in centrifuge at full speed for 90 sec to dry off all water and ethanol.
Label new Microcentrifuge tubes and put columns in them.
Elute them by adding 50uL of water down the middle of the column (don't let it stick to the sides).
Spin in centrifuge at top speed for 30 seconds.
Take out columns and cap the tubes.
Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.